Site-directed mutagenesis is an important procedure in studies of gene expression and protein structure/function relationships. A variety of protocols have been developed to mutate specific bases in plasmid DNA, which employ oligonucleotide primers containing desired mutations flanked by bases complementary to target sequences (reviewed in Smith M. (1985) Ann. Rev. Genet. 19:423-462). The primers are usually extended in vitro, although Mandecki (Proc. Natl. Acad. Sci. U.S.A. (1986) 83:7177-7181) has described a procedure in which the primers are presumably extended in vivo following transformation of a bacterial host. In the absence of mismatch-repair, DNA replication of heteroduplex DNA is expected to yield mutant products at frequencies of 50% or less. Since actual yields are often much lower than this theoretical maximum, strategies have been developed to select for products derived from the mutant strand. For example, a strategy has been developed by which circular single-stranded DNA (ssDNA) with several uracil bases incorporated in place of thymidine (produced in dut.sup.-, ung.sup.- E. Coli strains) is used as a template for primer-directed synthesis of a strand carrying the desired mutation. (Kunkel et al. (1987) Meth. Enzymol 154:367-382; Kunkel (1985) Proc. Natl. Acad. Sci. U.S.A. 82:488-492). This DNA is transformed into an ung.sup.+ strain which degrades the uracil-containing template strand, yielding mutant products at frequencies as high as 98%.
Another strategy is to use two mutagenic primers, one carrying the desired mutation, and the second that reverts a mutation in a selectable marker, such as the ampicillin-resistance (amp.sup.r) gene (Lewis et al. (1990) Nucleic Acids Res. 18:3439-3443), or gene IV from M13 (Carter (1987) Meth. Enzymol. 54:382-403). The two primers direct synthesis of a second strand carrying the reverted selectable marker and the desired mutation. Subsequent selection for the reverted marker yields products carrying the desired mutation at frequencies of about 80%. An analogous strategy couples the primer carrying the desired mutation to a primer that destroys a site recognized by the host restriction/modification system (Carter, ibid.). This strategy permits the efficient recovery of mutant plasmids resistant to the host restriction system. These coupled-primer systems benefit from the use of T4 DNA polymerase for primer extension, rather than the Klenow fragment of E. coli DNA polymerase, since the T4 enzyme does not displace the mutagenic primers (Masumune et al. (1971) J. Biol. Chem. 246:2692-2701; Nossal (1974) J. Biol. Chem. 249:5668-5676). Efficiency is also increased if mismatch repair-defective (mut S) hosts are used, increasing the probability that the reverted selectable marker and the desired mutation cosegregate during the first round of DNA replication (Zell et al. (1987) EMBO J. 6:1809-1815).
These specialized mutagenesis procedures impose several requirements. The dut.sup.- /ung.sup.- system (Kunkel, ibid.) requires that the target plasmid carry an fl origin of replication, the transformation of the target plasmid into an F, dut.sup.-, ung.sup.- host, and the preparation of circular ssDNA templates. Target plasmids must be derivatives of M13, or they must carry fl origins ("phagemids"). ssDNA is produced from phagemids by propagation in the presence of helper phage. Systems that employ revertible selectable markers require subcloning the target DNA into a specialized plasmid vector, and can require specific host strains to permit selection of the reverted marker (Carter, ibid.), or for the production of circular ssDNA templates (Lewis et al., ibid.). If multiple mutations are to be introduced sequentially, revertible marker systems usually require the reintroduction of target DNA fragments into the parent vector carrying the defective marker.